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Construction method of a kind of xylanase hybrid enzyme engineering strain

A construction method, xylanase technology, applied in the field of construction of xylanase hybrid enzyme engineering strains, to achieve the effect of increasing the optimum temperature

Active Publication Date: 2019-03-01
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, there are relatively few studies on improving the stability of xylanase through glycosylation modification, so there is some room for exploration

Method used

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  • Construction method of a kind of xylanase hybrid enzyme engineering strain
  • Construction method of a kind of xylanase hybrid enzyme engineering strain
  • Construction method of a kind of xylanase hybrid enzyme engineering strain

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Embodiment Construction

[0022] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0023] 1 Materials and methods

[0024] 1.1 Strains, plasmids and reagents

[0025] Aspergillus niger (Aspergillus niger) XZ-3S, Escherichia coli (Escherichia coli) JM109, DH5α were preserved by the author's laboratory; eukaryotic expression plasmid pPIC9K and Pichia pastoris (Pichia pastoris) GS115 were purchased from Invitrogen. DNA polymerase, restriction endonuclease and ligase were purchased from TaKaRa Company; ampicillin (Amp) was purchased from Sangon Company; gel recovery kit and plasmid extraction kit were purchased from Sangon Company; Ethidium bromide was purchased from Amresco Company; yeast powder, peptone and agarose were purchased from BBI Company; G418 and amino-free yeast nitrogen source (YNB) were purchased from Amresco Company; birch xylan was purchased from Sigma Company; other biochemical reagents were d...

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Abstract

The invention discloses a construction method of a xylanase hybrid enzyme engineering strain. First, the homology of the xylanase XynZF-2 gene sequence and the heat-resistant xylanase EvXyn11 gene sequence is compared by using bioinformatics, Replace the N-terminal 48 amino acids of XynZF-2 with 34 amino acids at the N-terminal of xylanase EvXyn11 by N-terminal substitution, and introduce aromatic amino acids P9Y and H14F, and then continue to introduce disulfide at the C-terminal of XynZF-2 enzyme Hydrophobic amino acids K164M, G166A, N160I, V111A, and G109A were introduced into the Cys38‑Cys191, α‑helix, and cord regions, respectively, and finally the glycosylation modification sites E42N and F17S were introduced to construct a multi-site mutant hybrid enzyme XynZL. The thermostability of the synthase XynZL has been improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing a xylanase hybrid enzyme engineering strain. Background technique [0002] Xylanase (EC 3.2.1.8) is a general term for a class of multifunctional enzymes that catalyze the hydrolysis of xylan. Xylanase is widely distributed in nature, such as animals, plants and microorganisms, and has a wide variety of applications. At present, there are many reports on xylanases derived from microorganisms, and the most studied are xylanases derived from bacteria and fungi. Among them, bacteria can produce alkaline and acidic xylanases, while fungi can produce alkaline xylanases. At present, xylanase is mainly produced by fermentation of microorganisms such as fungi and bacteria. [0003] With the continuous development of biotechnology, the research and development of xylanase has attracted much attention. In terms of industrial application, xylanase has great potential...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/56C12N15/81C12R1/84
CPCC12N9/2482C12Y302/01008
Inventor 周晨妍刘振华王燕王拥涛李同彪朱新术
Owner XINXIANG MEDICAL UNIV
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